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Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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A perforated patch recording method was used to
determine the effects of genistein (Gen), a protein tyrosine kinase
(PTK) inhibitor, on basal L-type
Ca2+ current
(ICa,L) in
feline atrial myocytes. Gen (50 µM) elicited biphasic changes in
ICa,L: an initial
inhibition (
55 ± 4%; phase 1) followed by a secondary stimulation (34 ± 9%;
phase 2) of
ICa,L. Withdrawal
of Gen elicited a further potentiation of
ICa,L (152 ± 19%; phase 3) above control
(n = 46). In general,
phase 1 inhibition and
phase 3 potentiation varied directly
with Gen concentration, and phase 2 stimulation exhibited biphasic concentration-dependent changes compared
with control. When cells were dialyzed using a ruptured patch recording
method, Gen elicited only inhibition of
ICa,L;
phases 2 and
3 were abolished. Vanadate (1 mM), an
inhibitor of protein tyrosine phosphatase, abolished both Gen-induced
inhibition and stimulation of
ICa,L. Daidzein
(50 µM), a weakly active analog of Gen, exerted no significant
effects on ICa,L,
and withdrawal of daidzein failed to potentiate
ICa,L. In a few
cells, Gen elicited a prominent vanadate-sensitive stimulation of
ICa,L in the
absence of any significant inhibition of
ICa,L.
Gen-induced changes in ICa,L were
unaffected by either 100 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM) or 1 µM ryanodine, agents that
alter intracellular Ca2+; 4 µM
H-89 or 50 µM Rp diastereomer of adenosine
3',5'-monophosphothioate (RP-cAMPS), inhibitors
of protein kinase A (PKA); 0.1 µM calphostin C or 2 µM
chelerythrine, inhibitors of protein kinase C (PKC); or 100 µM
NG-monomethyl-L-arginine
(L-NMMA), an inhibitor of nitric oxide (NO) synthase. We
conclude that in feline atrial myocytes, Gen acts via membrane-bound
PTKs to inhibit
ICa,L and via
cytosolic PTKs to stimulate
ICa,L.
Gen-induced changes in
ICa,L are not related to changes in intracellular
Ca2+ or to secondary interactions
with either PKA, PKC, or NO signaling pathways. These results indicate
that in atrial myocytes
ICa,L is
regulated by two independent and competing PTK signaling mechanisms.
electrophysiology; cardiac; vanadate; daidzein; perforated patch
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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PROTEIN TYROSINE KINASE (PTK) activity is a major
signaling mechanism that mediates the actions of a wide variety of
hormones and neurotransmitters, as well as multiple cellular processes governing cell growth and differentiation (33). PTK signaling also
plays an important role in the regulation of ion channel conductances
(28). Genistein (Gen) is an isoflavone that specifically inhibits PTK
activities (1) and is widely used as a pharmacological tool to
investigate the role of PTK signaling in a variety of systems (32). For
instance, the effects of Gen have implicated PTK signaling in the
regulation of K+ currents (15),
cardiac Cl
currents (27,
30, 31), Ca2+ mobilization in
smooth muscle (11, 24), L-type
Ca2+ current
(ICa,L) in
myometrial (20), vascular smooth muscle (15, 39), and pituitary cells
(2), and in activation of the
Ca2+-release activated current
that is elicited by depletion of intracellular Ca2+ stores in epithelial cells
(25). Less is known, however, about the potential role of PTK signaling
in the regulation of cardiac ICa,L. In
ventricular myocytes, Gen elicits inhibition of
ICa,L, although
the role of PTK signaling in this response was equivocal (4, 40). To
the best of our knowledge, no comparable studies are available in
cardiac atrial muscle. Therefore, in the present study, we sought to
examine the effects of Gen to gain insight into possible PTK signaling
mechanisms that may regulate
ICa,L in atrial
myocytes. The present results indicate that Gen exerts inhibitory and
stimulatory effects on atrial
ICa,L that are
both mediated via inhibition of PTK signaling mechanisms. Part of this work has been presented in abstract form (37).
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Details of the isolation and recording methods have been published previously (35). Adult cats of either sex were anesthetized with pentobarbital sodium (70 mg/kg ip). Hearts were perfused via a Langendorff apparatus with a bicarbonate-buffered Tyrode solution for ~5 min followed by perfusion with a nominally Ca2+-free Tyrode solution. After 5 min, the perfusion was switched to a low-Ca2+ (36 µM) Tyrode solution containing 0.06% collagenase (type II, Worthington Biochemical) for 30-40 min. After collagenase perfusion, both atria were cut into small pieces and agitated in fresh collagenase and 0.01% protease. Experiments were performed on either right or left atrial cells, with no discernible differences in responses. Cells studied were isolated on the morning of each experiment.
Cells used for study were transferred to a small tissue bath on the
stage of an inverted microscope (Nikon Diaphot) and superfused with a
modified Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 1.0 MgCl2, 2.0 CaCl2, 5 HEPES, and 11 glucose and
titrated with NaOH to a pH of 7.4. Solution was perfused through a
small (0.3 ml) chamber by gravity at ~5 ml/min. The system required ~20 s to completely exchange the bath contents. All experiments were
performed at 35 ± 1°C. Cells selected for study were elongated and quiescent. Ionic currents were recorded using a nystatin-perforated patch (14) whole cell recording method (12). This method minimizes dialysis of intracellular contents with the internal pipette solution, thereby maintaining physiological buffering of intracellular
Ca2+ and second messenger
signaling pathways. Nystatin was dissolved in DMSO at a concentration
of 50 mg/ml and then added to the internal pipette solution to yield a
final nystatin concentration of 150 µg/ml. The pipette solution
containing nystatin was strongly sonicated before use. The internal
pipette solution contained (in mM) 100 cesium glutamate, 40 CsCl, 1.0 MgCl2, 4 Na2ATP, 0.5 EGTA, and 5 HEPES and
was titrated with CsOH to a pH of 7.2. To record
ICa,L, K+ currents were blocked by
Cs+ in the internal pipette
solution and 20 mM CsCl in the external solution. Addition of CsCl to
the external solution was not osmotically compensated. In some
experiments, a ruptured patch recording method was used to dialyze
intracellular contents with internal pipette solution. When the
ruptured patch method was used, the internal pipette solution was the
same as indicated above except that EGTA concentration was 10 mM and
CaCl2 concentration was 0.44 mM
(pCa 7). In the ruptured patch configuration, the liquid junction
potential (10 mV) measured between the internal pipette and bath
solutions was subtracted from all voltage measurements. A single
suction pipette recorded ionic currents (switch clamp) using an
Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) in both
perforated and ruptured patch recording configurations. The switch
clamp precludes the need to compensate for series resistance. When
filled with internal solution, the pipette tip resistance was ~3
M
. In the perforated patch configuration, access resistance was
~15-20 M, and in the ruptured patch configuration, access
resistance was ~10 M. The sampling rate of the switch clamp was
~10-12 kHz, and a second oscilloscope was used to monitor the
duty cycle to ensure that the voltage transient settled between cycles.
Computer software (pCLAMP 6.2; Axon Instruments) was used to deliver
voltage protocols and to acquire and analyze data. In addition, all
signals were digitally recorded on videocassette recorder tape.
Generally, ICa,L
was activated by clamp steps from a holding potential of 40 to 0 mV for 200 ms every 5 s. This voltage protocol avoids activation of
fast Na+ and T-type
Ca2+ currents. In the experiments
using a rupture patch recording method, rundown of
ICa,L stabilized
within ~6 min of rupturing the patch. In general, the extent of
rundown was variable from cell to cell. In three cells, rundown
decreased peak
ICa,L by 48 ± 6%. The effects of Gen on
ICa,L were
recorded after rundown of
ICa,L stabilized.
Peak ICa,L was
measured with respect to steady-state current and was not compensated
for leak currents. The time constant of
ICa,L
inactivation was best fit as a single exponential using Clampfit
(pCLAMP 6.2). Statistical significance of paired and unpaired data were
determined by Student's t-test at
P values <0.05. Data are expressed
as means ± SE. The animal procedures used in this study were in
accordance with the guidelines of the Animal Care and Use Committee of
Loyola University Medical Center.
Drugs and chemicals used in this study include Gen, daidzein, sodium orthovanadate, and 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM) (Sigma Chemical); H-89 (N-[2-p-bromocinnamylamino]-5-isoquinoline sulfonamide) (Seikagaku America); the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) and chelerythrine chloride (LC Laboratories); ryanodine (Progressive Agri-Systems); and calphostin C (Kamiya Biomedical). Gen and H-89 were prepared as stock solutions in DMSO. Final concentration of DMSO was 0.05% and had no effect on basal ICa,L. Sodium orthovanadate was prepared at its final concentration in buffered Tyrode solution.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Figure 1,
A-C,
shows the effects of 50 µM Gen on selected original records of basal
ICa,L
(A), consecutive measurements of peak ICa,L
amplitude (B), and mean percent
changes in peak
ICa,L (C). Exposure to Gen elicited an
initial inhibition of
ICa,L (Fig. 1Aa, phase
1; Ba) that was
followed by stimulation of
ICa,L above control levels (Fig. 1Ab,
phase 2;
Bb). Phase
2 stimulation of ICa,L remained
relatively constant during exposure to Gen (Fig. 1Bb). After 2 min of exposure,
removal of Gen elicited a prominent additional increase or potentiation
in ICa,L
amplitude (Fig. 1Ac, phase 3;
Bc) and then returned
ICa,L to control
levels. Gen had no effect on the holding current. In a total of 46 cells (Fig. 1C), Gen-induced changes
in peak ICa,L
elicited during phase 1 inhibition,
phase 2 stimulation, and
phase 3 potentiation were 55 ± 4% (P < 0.001), 34 ± 9%
(P < 0.01), and 152 ± 19%
(P < 0.001), respectively, compared
with control. Phase 3 potentiation
represents an additional increase of 118% above the level of
phase 2 stimulation. Measured in 14 cells, from the onset of exposure to Gen, phase 1 inhibition and phase
2 stimulation reached their peaks by 30 ± 5 and 69 ± 5 s, respectively. From the withdrawal of Gen,
phase 3 potentiation of
ICa,L reached its
peak at 40 ± 6 s, and
ICa,L required
118 ± 11 s to return to control levels. These results indicate that
exposure to Gen elicits a biphasic effect on
ICa,L amplitude,
an initial inhibition followed by a secondary stimulation. The biphasic
nature and the fact that the stimulatory component is potentiated when
Gen is withdrawn suggests that Gen is acting via two separate and
competing signaling mechanisms. Because phase 2 stimulation is the net result of two opposing
effects, its magnitude was more variable than either
phase 1 or phase
3. Hence, the amplitude of
ICa,L during
phase 2 was usually, but not always,
larger than control
ICa,L. We have
adopted the terminology phase 2 stimulation because after phase 1 inhibition, ICa,L
amplitude invariably increased with time during continued exposure to
Gen, even though it did not always become larger than control
ICa,L.
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Next, we determined a dose-response relationship for Gen-induced
changes in peak
ICa,L. The graphs
in Fig. 2,
A-C,
show the phase 1 (A), phase
2 (B), and
phase 3 (C) responses to different Gen
concentrations ranging from 0.1 to 100 µM. In general,
phase 1 inhibition (Fig.
2A) was directly related to the Gen
concentration, phase 2 stimulation
showed biphasic changes in relation to control, and
phase 3 potentiation (Fig.
2C) increased with Gen
concentrations. More specifically, Gen concentrations <1 µM had no
significant effects on
ICa,L. Exposure
to 1 µM Gen failed to elicit significant phase
1 or phase 2 changes,
although withdrawal of 1 µM Gen elicited a significant
phase 3 increase in
ICa,L. At 10 µM
Gen, phase 1 inhibition became
significant and phase 3 potentiation
increased. At 100 µM Gen, phase 1 inhibition and phase 3 potentiation
were further increased and phase 2 stimulation became significant. The direction and amplitude of
phase 2 depended on the net
Gen-induced effects on
ICa,L. At Gen
concentrations 
10 µM, the net stimulatory effect resulted in
phase 2 stimulation above control
levels. At 100 µM Gen, the more prominent inhibitory effect resulted
in phase 2 stimulation that did not
exceed control levels. These results indicate that Gen induces a
dose-dependent inhibition and stimulation of
ICa,L and that
phase 2 was biphasic as a result of
the net effect of these two opposing responses. It is worth mentioning that because of the apparent competition between the stimulatory and
inhibitory components, the response to each Gen concentration and
therefore the sensitivity of each component is probably underestimated in these experiments.
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If one of the component changes in
ICa,L induced by
Gen is related to a soluble cytosolic factor(s), then dialysis of
intracellular contents may provide a means of separating the two
effects of Gen. We therefore tested 50 µM Gen while recording
ICa,L using a
ruptured patch, rather than a perforated patch, recording method. Figure 3,
A-C,
shows the effect of Gen on selected original
ICa,L traces
(A), consecutive measurements of
peak ICa,L
(B), and mean percent changes in
peak ICa,L
(C). In these experiments, Gen was tested after the rundown of
ICa,L had
stabilized. Gen elicited an initial phase
1 inhibition of 85% (Fig. 3,
Ab and
Bb) that diminished only slightly
during continued exposure to Gen (Fig. 3,
Ac and
Bc). As a result, during
phase 2,
ICa,L amplitude
still was inhibited significantly compared with control. Withdrawal of
Gen returned
ICa,L to control
levels with no phase 3 potentiation (Fig. 3, Ad and
Bd). In other words, withdrawal of
Gen simply removed the inhibitory component. In a total of six cells
tested (Fig. 3C), Gen-induced
changes during phase 1 and
"phase 2" were 68 ± 9 and 57 ± 8%, respectively, and withdrawal of Gen simply returned ICa,L to
baseline. In two additional cells when Gen was tested within 3 min of
rupturing the membrane patch, i.e., before rundown of
ICa,L had
stabilized, the stimulatory components were still evident. These
results indicate that dialysis of the cell interior eliminated the
phase 2 stimulation and
phase 3 potentiation typically induced
by Gen when recordings are performed using a perforated patch method.
In addition, they indicate that both stimulatory phases
(2 and
3) induced by Gen are due to the
same mechanism that involves a soluble cytosolic factor(s). On the other hand, the inhibitory effects of Gen result from a membrane-bound mechanism.
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To assess the relative specificity of Gen action, we examined the
effects of 50 µM daidzein, a weakly active analog of Gen (1), on
ICa,L. The
effects of 50 µM Gen and 50 µM daidzein were tested in the same
atrial myocytes to ensure that cells exhibited a typical response to
Gen before being tested with daidzein. In a total of four cells
studied, Gen elicited phase 1 inhibition (24 ± 3%), phase
2 stimulation (212 ± 52%), and
phase 3 potentiation (303 ± 43%)
of ICa,L.
Daidzein exerted no significant inhibitory or stimulatory effects on
ICa,L, and
withdrawal of daidzein had no effect on
ICa,L.
Effects elicited by Gen-induced inhibition of PTK activities depend on
intact protein tyrosine phosphatase (PTPase) activity to
dephosphorylate tyrosine residues. Vanadate (Van) enhances tyrosine
phosphorylation by inhibiting PTPase activities (32, 34) and thereby
can prevent the effects of Gen that are mediated by PTK inhibition. As
shown Fig. 4,
A and
B, we therefore tested Gen in the
absence and then presence of 1 mM Van to determine whether the
inhibitory and/or stimulatory effects of Gen are mediated via
inhibition of PTK activities. Cells were exposed to Van for 4 min
between the first and second exposure to Gen. The graph in Fig.
4A shows consecutive measurements of
peak ICa,L
obtained from a single atrial myocyte. Under control conditions, 50 µM Gen elicited phase 1 inhibition
(33%), a prominent phase 2 stimulation (110%), and phase 3 potentiation (143%) of
ICa,L. Van alone
decreased ICa,L
by ~8%. It should be noted, however, that after the initial exposure
to Gen, ICa,L
stabilized at a level somewhat higher than control. As a result, Van
simply decreased
ICa,L back to the
control level, probably by inhibiting a residual stimulatory effect of Gen. In the presence of Van, Gen-induced phase
1 inhibition was abolished, phase
2 stimulation was significantly attenuated, and phase 3 potentiation of
ICa,L was
abolished. As summarized in Fig. 4B,
in the five cells studied, under control conditions Gen induced
phase 1 inhibition (30 ± 5%), phase 2 stimulation (100 ± 15%), and phase 3 potentiation (134 ± 34%) of
ICa,L. In the presence of Van, Gen-induced changes in phases
1, 2, and
3 were 3 ± 2, 18 ± 5, and 17 ± 6%, respectively (P < 0.05). In two control cells, two consecutive exposures to Gen separated
by a 4-min period elicited typical changes in
ICa,L that were
not significantly different from one another (data not shown). On the
basis of the mean value for the five cells, Van alone had no
significant effect on peak
ICa,L amplitude
(2 ± 6%). This mean value, however, was obtained from
individual experiments where Van elicited variable changes in
ICa,L among the
different cells tested. Specifically, Van decreased
ICa,L in three
cells (8, 15, and 6%), increased ICa,L in one cell
(22%), and in a fifth cell, Van had no effect compared with control.
Van also elicited small and variable changes in
ICa,L
inactivation that were not significant (2 ± 4%;
n = 5). Because Van always was
administered after an initial exposure to Gen, it seems likely that the
variable effects of Van alone were influenced by the residual effects
resulting from the initial exposure to Gen (see Fig.
4A). Nevertheless, the present
results indicate that regardless of the effects of Van on
ICa,L, Van
blocked both the inhibitory and stimulatory effects of Gen.
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Although Van is a potent inhibitor of PTPase activity (32, 34), it has
been reported to exert other effects as well (3, 10, 23). For example,
in relation to the present study, Van may inhibit serine/threonine
phosphatase activity and/or act as a
Pi analog, thereby interfering
with cellular mechanisms involving serine/threonine phosphorylation or
ATP hydrolysis. To access these possibilities, we tested Van in cells
where ICa,L had
been prestimulated by isoproterenol (Iso) at a concentration (0.02 µM) that submaximally stimulates
ICa,L. If Van
significantly inhibits serine/threonine phosphatase activity or
interferes with ATP hydrolysis, it should significantly alter
-adrenergic stimulation of
ICa,L. Figure
5 shows a typical experiment in which Iso
alone increased peak
ICa,L by 177%.
The addition of 1 mM Van to the Iso-stimulated cell had no effect on
peak ICa,L
ampitude. When Iso was withdrawn, leaving the cell in the presence of
Van, ICa,L
amplitude returned to within 28% of control. This return of
ICa,L toward
control would not be expected if Van acted to significantly inhibit
serine/threonine phosphatase activity or stabilize the transition state
for phosphate tranfer. Removal of Van returned
ICa,L back to
control. In a total of three cells, the effects of Iso on
ICa,L amplitude
in the absence and presence of Van were 123 ± 34 and 120 ± 32%, respectively, and the withdrawal of Iso returned
ICa,L amplitude
to within 30% of control. These findings provide support for our
interpretation that under the present experimental conditions Van is
blocking the effects of Gen on
ICa,L primarily
via inhibition of PTPase activities.
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In a few cells studied, Gen elicited an atypical effect on ICa,L that may provide insight into the underlying mechanisms of Gen action. Figure 6 shows selected recordings of ICa,L (A) and consecutive measurements of peak ICa,L (B) recorded from the same atrial myocyte. Exposure to 50 µM Gen failed to induce phase 1 inhibition of ICa,L and instead elicited only marked stimulation of ICa,L (109%) above control. Withdrawal of Gen induced a small potentiation of ICa,L (129%) above control, an additional increase of 20%. These Gen-induced stimulatory effects on ICa,L were blocked by 1 mM Van (data not shown). In a total of five cells that showed this type of response, Gen stimulated ICa,L by 123 ± 35%, and withdrawal of Gen potentiated ICa,L to 150 ± 42% above control, an additional increase of 27%. In the presence of Van, Gen-induced stimulation (7 ± 3%) and potentiation (19 ± 2%) were abolished (n = 2). This type of response was selected to demonstrate that Gen could elicit a prominent stimulation of ICa,L via PTK signaling in the absence of any significant inhibition of ICa,L.
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PTK signaling may interact with protein kinase A (PKA) or protein kinase C (PKC) signaling mechanisms that regulate ICa,L. Therefore, in Fig. 7, A and B, we tested the effect of H-89, an inhibitor of PKA (5), and calphostin C, a specific inhibitor of PKC (19), respectively, on Gen-induced changes in ICa,L. Previous work from this laboratory has shown that H-89 (35) and calphostin C (36) are effective in blocking PKA- and PKC-mediated regulation of ion channels, respectively, in these atrial myocytes. Exposure to 50 µM Gen in the absence and presence of 4 µM H-89 showed no differences in Gen-induced changes in ICa,L (Fig. 7A). Likewise, cells exposed to 50 µM Gen in the absence and then presence of 0.1 µM calphostin C showed no differences in Gen-induced changes in ICa,L (Fig. 7B). In three additional cells, 50 µM Rp-cAMPS, a more specific inhibitor of PKA (6), failed to affect either the inhibition or stimulation of ICa,L induced by Gen (data not shown). Previous work has shown that superfusion of cat atrial myocytes with Rp-cAMPS blocks cAMP-mediated stimulation of ICa,L (35). Moreover, 2 µM chelerythrine, another specific PKC inhibitor (13), had no significant effect on Gen-induced changes in ICa,L (n = 3) (data not shown). The present results, therefore, indicate that the effects of Gen on basal ICa,L are not mediated via secondary interactions between PTK signaling and PKA or PKC signaling mechanisms.
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Nitric oxide (NO) also can modulate atrial
ICa,L amplitude
via second messenger cGMP signaling mechanisms (17, 38). To determine
whether the effects of Gen may be mediated via NO signaling, Gen was
tested in the absence and presence of 100 µM
NG-monomethyl-L-arginine
(L-NMMA), an inhibitor of NO
synthase activity (18). Under control conditions, 50 µM Gen elicited
typical phase 1 (62 ± 3%),
phase 2 (43 ± 21%), and
phase 3 (55 ± 18%) changes in
ICa,L. After
recovery from Gen, exposure to
L-NMMA alone slightly decreased
ICa,L (7 ± 2%). In the presence of
L-NMMA, Gen-induced changes in
ICa,L during
phase 1 (55 ± 18%),
phase 2 (68 ± 25%), and
phase 3 (78 ± 34%) were not
significantly different from control responses.
Because these experiments are performed with the perforated patch
method, it is possible that secondary alterations in intracellular Ca2+ may contribute to the effects
of Gen on ICa,L.
We therefore tested the effects of Gen in cells exposed to 100 µM
BAPTA-AM, a cell-permeable Ca2+
chelator. BAPTA-AM abolished visible contractile activity associated with activation of
ICa,L, slowed
ICa,L
inactivation (18 ± 7%), and increased peak
ICa,L amplitude
(16 ± 4%). These changes are consistent with the effect of BAPTA
to bind intracellular Ca2+ and
reduce Ca2+ concentration close to
the channel. In a total of seven cells tested, in the presence of
BAPTA-AM, 50 µM Gen elicited a typical phase
1 inhibition (28 ± 4%),
phase 2 stimulation (+26 ± 14%), and phase 3 potentiation (+69 ± 20%) of ICa,L.
In three additional cells, we found that 1 µM ryanodine, an alkaloid
that depletes intracellular Ca2+
stores (7) and abolished visible contractile activity, had no effect on
Gen-induced changes in
ICa,L (data not
shown). Another consideration is that cells held at 40 mV are
close to the theoretical equilibrium potential for
Na+/Ca2+
exchange. This could inhibit Ca2+
efflux, allowing intracellular
Ca2+ to increase. We therefore
performed a voltage-clamp protocol where the cell was held at 80
mV between pulses and then ramped to 40 mV immediately before
activation of
ICa,L. With the
use of this protocol in a total of five cells, Gen elicited typical phase 1 inhibition (43 ± 9%), phase 2 stimulation (+30 ± 7%), and phase 3 potentiation (+134 ± 40%) of
ICa,L. Together,
these results indicate that changes in intracellular free
Ca2+ or intracellular
Ca2+ release from internal stores
are not factors in either the inhibitory or stimulatory effects of Gen
on ICa,L.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The present study indicates that Gen elicits a biphasic effect on
ICa,L that is
mediated via two competing PTK signaling mechanisms: an initial
inhibitory component that is overcome by a secondary stimulatory
component. These two components appear to result from independent
signaling mechanisms. Thus, during intracellular dialysis, Gen could
elicit the inhibitory component in the absence of the stimulatory
response. Moreover, in some cells, a prominent stimulatory response
could be elicited by Gen in the absence of any significant inhibitory
response. Competition between these two opposing mechanisms also is
evident in the variable phase 2 changes in ICa,L
induced by any given concentration of Gen, and in the biphasic changes in phase 2 that were dose dependent.
Furthermore, when the stimulatory component was eliminated by cell
dialysis, phase 1 inhibition changed
from a transient to a sustained response. In addition, cell dialysis
eliminated both phase 2 and
3 stimulatory components and showed
that the withdrawal of Gen simply removed a sustained inhibitory
component, returning
ICa,L to
baseline. These results clearly indicate that the
phase 3 potentiation of
ICa,L typically elicited by withdrawal of Gen resulted from the rapid removal of an
inhibitory component competing with a more sustained stimulatory component. This also is consistent with the relatively small
potentiation of
ICa,L elicited by
withdrawal of Gen in the few cells in which Gen failed to elicit an
inhibitory component. This last observation raises an interesting
point; although Gen failed to elicit any noticeable inhibition of
ICa,L, withdrawal
of Gen still induced a small potentiation of
ICa,L. This
suggests that the relatively large stimulatory response of these
particular cells masked a smaller inhibitory effect of Gen and that
upon withdrawal of Gen the underlying inhibitory signal induced by Gen
was removed, resulting in a small potentiation of
ICa,L. The
present findings also indicate that the mechanisms underlying
Gen-induced inhibition of
ICa,L are more
rapid in onset and more rapidly removed than those underlying stimulation of
ICa,L. However,
at Gen concentrations 50 µM, the stimulatory component is more
potent and can overcome the inhibitory component. Therefore, at
relatively low Gen concentrations, the stimulatory component is able to
attenuate or even mask the inhibitory effect on
ICa,L.
These findings can be interpreted in terms of several different PTKs that could potentially be inhibited by Gen. PTKs are categorized into two general groups: receptor-operated and non-receptor-operated (33). Receptor-operated PTK activities are considered membrane bound, whereas non-receptor-operated PTK activities are soluble, cytosolic components that may be compartmentalized within the cell. Also, there are membrane-associated PTKs such as the Src (9) and Jak families (16) that can dissociate from the membrane once activated and affect cytosolic signaling mechanisms. Several findings support the idea that Gen-induced inhibition and stimulation of ICa,L are both mediated via inhibition of PTK activities. First, the effects of Gen could not be mimicked by daidzein, an analog of Gen but weak inhibitor of PTK activity. Moreover, the dose-response relationship suggests that the half-maximal inhibitory and stimulatory concentrations of Gen are ~50 µM, which is well within the concentration range for specific PTK inhibition (1). In addition, Van, a potent inhibitor of PTPase activities, essentially abolished both the inhibitory and stimulatory effects of Gen. As discussed earlier, Van can exert various nonspecific effects (3, 10, 23). The present results, however, suggest that under our experimental conditions, the ability of Van to block Gen-induced changes in ICa,L was due to inhibition of PTPase activities. Thus Van failed to affect Iso-induced stimulation of ICa,L. This finding would not be expected if Van acted nonspecifically as an inorganic phosphate analog or via inhibition of serine/threonine activity. Moreover, this last observation is consistent with the present findings that the effects of Gen are not mediated via PKA- or PKC-mediated signaling. Although Van probably does exert various effects, the most likely explanation of its actions in the present experiments is via inhibition of PTPase activities. Taken together, the present results suggest that Gen-induced inhibition of ICa,L is mediated via inhibition of membrane-bound or -associated PTKs, and Gen-induced stimulation of ICa,L is mediated via inhibition of cytosolic PTK activities.
As alluded to earlier, receptor-operated or membrane-associated PTKs also may interact with cytosolic PTKs to affect downstream signal transduction mechanisms that could stimulate ICa,L. Although this possibility is not excluded by these experiments, several of the present observations make it unlikely. First, prominent stimulatory responses could be elicited by Gen in the absence of any significant inhibitory effects on ICa,L. In addition, this mechanism is not consistent with the present finding that removal of the inhibitory component by withdrawal of Gen potentiated the stimulatory response. Moreover, Gen-induced changes in ICa,L were not mediated via secondary interactions with PKA or PKC pathways or regulated by intracellular Ca2+ signaling. This makes it unlikely that certain PTK activities that are activated by intracellular Ca2+ and/or PKC activities, such as PYK2 (21), are involved in the effects of Gen. Likewise, other Ca2+-dependent signaling mechanisms such as activation of Ca2+/calmodulin-dependent protein kinase II (8) or Ca2+-dependent NO synthase (18) are probably not involved either. The latter statement is further supported by the present finding that inhibition of NO synthase activity by L-NMMA had little effect on Gen-induced changes in ICa,L. That intracellular Ca2+ does not play a role in the effects of Gen also makes it unlikely that intracellular dialysis eliminated the stimulatory component by buffering intracellular Ca2+. Of course, the present experiments are not exhaustive, and therefore, it is possible that other signaling pathways not examined in the present study and modulated by PTK signaling may contribute to the effects of Gen on ICa,L.
In contrast to the present findings, there are reports indicating that Gen may directly block membrane channels by a mechanism unrelated to PTK inhibition. For example, in vascular smooth muscle cells, Gen blocks K+ currents by a mechanism independent of ATP utilization and insensitive to inhibition by vanadate (29). In rat brain neurons, Gen inhibited Na+ influx and Na+ current, but daidzein also elicited similar inhibitory effects as Gen, albeit at higher concentrations than Gen (22). In cardiac ventricular myocytes, Gen and daidzein both elicited similar inhibition of ICa,L (4, 40). Clearly, these findings differ significantly from those presented here, where the effects of Gen on ICa,L were not mimicked by daidzein and were effectively blocked by vanadate.
An important aspect of the present study is that we used a perforated
patch recording method to study PTK-mediated regulation of
ICa,L. The
studies cited above each used whole cell ruptured patch methods. The
importance of maintaining the intracellular milieu is evident from the
present experiments where cell dialysis using the ruptured patch method
abolished the stimulatory component, leaving only Gen-induced
inhibition of
ICa,L. This
finding may have bearing on the interpretation of studies in
ventricular myocytes where recordings performed with the ruptured patch
method indicated that Gen elicits only inhibition of
ICa,L (4, 40).
This raises the question of whether the ruptured patch recording method
influenced the results obtained in ventricular myocytes and the
possibility that Gen may also elicit a stimulatory effect on
ICa,L in those cells. In fact, in cat ventricular myocytes, Gen elicited a
phase 1 inhibition (54 ± 6%), phase 2 stimulation (33 ± 4%), and phase 3 potentiation
(23 ± 8%) (n = 10), indicating
that the stimulatory components were present but significantly smaller
in magnitude than in cat atrial myocytes (37).
The present results suggest that Gen elicits both an inhibitory and stimulatory effect on ICa,L via dephosphorylation of different tyrosine residues that compete in the regulation of ICa,L. Others have reported that dual regulation of cardiac ICa,L also can be achieved by phosphorylation of serine/threonine residues where cGMP enhances cAMP/PKA activity to stimulate or protein kinase G activity to inhibit ICa,L (26, 35). In the present experiments, the net steady-state effect of Gen on ICa,L appears to be a balance between two opposing PTK signaling mechanisms. These results therefore suggest that atrial muscle functions mediated by ICa,L may be strongly regulated by neurotransmitters or hormones that act via receptor-operated and/or non-receptor-operated PTK signaling mechanisms.
| |
ACKNOWLEDGEMENTS |
|---|
We thank the late Christine E. Rechenmacher for expert technical assistance with these studies. In addition, we thank Drs. Pamela Lucchesi and Alan Samarel for very helpful discussions regarding these experiments.
| |
FOOTNOTES |
|---|
This work was support by National Heart, Lung, and Blood Institute Grant HL-27652.
Address for reprint requests: S. L. Lipsius, Dept. of Physiology, Loyola University Medical Center, 2160 S. First Ave., Maywood, IL 60153.
Received 9 October 1997; accepted in final form 2 April 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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) in
ICa,L during
phase 2.
C: percent stimulation of
ICa,L during
phase 3. Exposure to Gen elicited a
dose-dependent inhibition and withdrawal of Gen elicited a
dose-dependent potentiation of
ICa,L. Changes in
phase 2 were biphasic and depended on
net effect of Gen-induced inhibition and stimulation of
ICa,L. Effect of
each Gen concentration on phases
1-3 was statistically analyzed in relation to its
own control; n = 9 or 10 cells at each
Gen concentration. * P < 0.05. # P < 0.01.
